Process of recovering petones and hematin from blood



UNITED STATES PATENT OFFICE.

V ELMORE E. BUTTERFIELD, OF FOREST HILLS, NEW YORK.

PROCESS OF RECOVERING PEPTONES AND HEMATIN FROM BLOOD.

No Drawing.

To all u; hom it may concern Be it known that I, ELMORE E. BUTTnR- FIELD, a citizen of the United States, residing at Forest Hills, in the county of Queens and State of New York, have invented certain new and useful Improvements in Processes of Recovering Peptones and Hematin from Blood, of which the following is a specification.

My invention relates to a process for the separation and quantitative recovery of peptones, such as globin and serum proteins in a state of partial hydrolysis, and also of hematin fromblood.

The process consists in thoroughly laking blood with hot water and a mineral acid to which is added pepsin. A detergent, such as fullers earth, etc., or a suitably freshly formed precipitate, which may be roduced in presence of the blood, is then ad ed to the mixture and it is again heated. It is then filtered while hot and the filtrate after neutralization of the acid is evaporated to yield peptone.

The residue on the filters is dissolved 1n dilute caustic soda and filtered and the hematin in the filtrate is reprecipitated by dilute acid and separated by filtration.

A specific example of the process is'as follows:

(1) Heat 1900 pounds of H O+20 ounds of HCl of 1.19 gravity to 4552 .2. (2) add 500 pounds of blood (whole or defibrinated but in liquid state) (3) mix thoroughly: (4) add 1.5 pounds of(pepsin (1:3000) preferably dissolved in H (5) digest for 12 hours at 45-50 0.: (6) bring to boil, add 20 pounds of detergent or one pound of sodium nucleinate and boil for 5 minutes: (7) filter while hot near 212 F. as possible: (8) evaporate filtrate, first neutralizing to absence of free mineral acid which requires about 5 pounds of NaOH. Filtrate neutralized and evaporated yields about 100 pounds or 20% of commercial peptone or' practically theoretical yields from 500 pounds of blood, 13% derived from hemoglobin, et%5% derived from serum proteins and the balance out of a theoretically possible 20% due to NaCl of neutralization: (9) residue on filters dissolved in dilute NaOH, filtered, and hematin in filtrate reprecipitated by dilute acid, and separated by filtration.

Specification of Letters Patent.

Application filed February 14, 1920.

Patented Jan. 17, 1922.

Serial No. 358,768.

The mechanism of the process is as follows:'

The blood is added to hot dilute HCl in the given proportions, (1) to effect a solution of the hemoglobin from the corpuscular elements, otherwise pepsin will not act particularly on intact colpuscles, (2) to convert the dissolved hemoglobin as well as the serum proteins into acid albumin which is readily attacked by the pepsin.

The whole reaction proceeds with all of the elements in solution from the beginning and in fairly dilute solution to furnish a high reaction velocity.

The hematin gradually separates out as it is split off from the globin fraction of the hemoglobin molecule, the separation beginning as early as four hours and sometimes being complete within siX to eight hours. The separation is greatly facilitated by boiling at the end of the reaction period which throws out practically all of the hematin with traces of undigested and co agulable protein: the detergent or precipitate of nucleic acid in statu nascendi removes traces of hematin and other coloring matter from the solution.

The following factors must be considered:

Dilution: 1 part of blood by volume to 4: volumes of H 0, i. e. a dilution of 1:5 is recommended. A dilution of 1:3 (1 part blood I- 2 parts water) is feasible but is workable with difficulty owing to bulkiness of hematin residue and viscosity of solution resulting in poor separation, low yields of peptone and slow filtration. Dilutions of less than 1 :8 (1 part of blood 1.75 H O) present increasing difficulties and when 1:2 (1 part blood 1 part E o) is reached there is little or no attacking of the globin molecule and only 7% peptone out of a possible 20% is recovered. Dilutions above 1 :5 are easily workable but necessitate the evaporation of large quantities of water and result in a product containing more salt and ash.

Acidity: A HOl concentration of 0.31% 0.35% is chosen rather than the theoretically accepted 0.15%0.2% because about one-third or more of the HCl is consumed in forming acid albumin and does not con tribute to acidity of the solution. If a concentration as low as 0.18% is used the filtrate will not turn congo, the yield is resharp but so much alkali is required v:tor.

5 neutralization that the resulting product Will contain or more salt and ash. Equivalent concentration of H SO may be used in which case a barium compound is usedit'or neutralization and clarification.

V Pepsin concentration; 1.5%by wei ht of the protein to be digested has been found sufficient for; 12 hours digestions In fact 1% is enough, the extra one-half of 1%being simply ayfactor ofsai'e ty. Witl'npro- 15 longationsof the tiine,-less than 1%in1ay. be

.used: (and s the time may i be: shortened by 111011 of vthe seruin proteins .andIhemoglobm ,--usingiinore than} 1.5%., 'I'IOWGYQI,ilk-0110 case vthe convenience and rapidity otfdlie:

process 1 suffer while 1D; the other; ucasea; the

20, cost is raised .proportionately.

Time of digestion: hiostall processcsior:

-, commerciahpeptonerequire lilloln fi'ztt) '72 hours.

gestioi is often complete in "SiX"l1OHIS,,LlSU

ally in eight hours and always; Within twelve hours.

The reaction nixture need not be boiled .as separation into a; clear l1qu1dand-,fiocculent masses occurs at160 E, but thehenia- .tin masses are-loose and spongy; {lIltlzfi-ltll- "tion is slow. fllie boiling effects a compact tion ofthe lieniatin and-the reaction mixture,

filters clearly "andirapidly. Sometimes ther separation takes place at the temperature of I tl1(5- reaction.=.(45 .509 ;C.),epart-icularly in .Cl1lllt6-SOlUt1011- =and; ion prolonged digestion,

Under the 5 conditions stated :.thedidilute solution; zofnalkali rand; the liema.tin

but boiling is always essential torapid filtration.

' Detergent: The detergent or i'reshly formed V precipitate removes traces. of dissolved hematm and other coloring matters, yielding an -r almost ;colorlessfiltrate; and-consequently a dry peptone with a minimum of color, Whiter thanpentone from fibrin or meat.

While I have described my inventionin detail, I desire ititorbe underst odrthat-the wthc= proteins of the; blood frouia-the heniatin which comprises digestiomof a diluteisoluwith it mineral acid zaiidapepsin a gglutinatiiig -,tl16=:l1611'l21l3l11 separating tl16-SOll1t1OR ioi 2: *The processscasfzclainied iii .z-claiin .1 in which the. heniatinic residue 1s, dissolved in a precipitated by a dilute acid.

3;.1'T'lie process;ass;Qlaimedtinmlaiin l in which i119;i ggllltllliltiOllils effected by adding a: detergent to the; dilute;solution, heat ing and then filtering;

a: 4'; illheyprocess as; clain edfliii claiin 2. in

:which; the; agglutination isieffected-by adding a detergent to;the-1 dilute solution lieae eing a d-11:11am tfi i erin 1 In testimony hereoif,;l;afii: my signature. 

